The Study Of The Serum From Mice With Traumatic Brain Injury And Limb Fractures On Osteoclast Differentiation

BackgroundOsteoblasts and osteoclasts are important cells that participate in fracturehealing process. Both bone forming by osteoblasts and bone absorbing by osteoclastsmake newly formed bone’s structure to meet the biomechanical requirements. Anyfactors that affecting osteoblasts or (and) osteoclasts, will influence the fracturehealing process.TBI patients with limb fractures had an increased amount of callus andaccelerated fracture healing. However, the exact mechanism is elusive. Researchesfocus on impact of serological changes on osteoblasts, while few researches paidattention to osteoclasts. Semaphorins3A（Sema3A）is an important signaling protein inthe nerve repair process, a study shows that the Sema3A has the promotion ofosteogenesis. Therefore, we speculate that Sema3A may changes in serum of TBI andfracture mice, and have an impact on osteoclasts. In this study, we analysis changes ofSema3A in serum from mice with TBI and femoral fracture, and observe in vitro cellculture of pre-osteoclasts with serum from mice with TBI and femoral fracture. Wehypothesize that Sema3A may increase the amount of callus and accelerate fracturehealing by inhibiting function of osteoclasts.ObjectiveTo establish a mouse model of traumatic brain injury combined with femoral fracture,and measure the change of Sema3A in serum from mice with TBI and femur fracture by ELISA, meanwhile, observe the effect of the serum from mice with TBI andfemoral fracture on osteoclasts’differentiation and function.Methods①Aweight-drop device was used to establish a traumatic brain injury in mice, in themeantime, transversely cut down the femur in order to establish a fracture model.Vital signs and pathological changes in the mouse after injury were observed, and a10-point neurological severity score (NSS) scale was used to evaluate the severity ofmouse’s brain injury.②The mice were divided into three groups: group1, TBI with femur fracture; group2, only TBI; group3, only fracture. After1d,3d,5d,1w and2w, blood wasobtained, followed by blood centrifugation.③ELISA was used to assay the changes of Sema3A and OPG in serum of the micein each group at each time point.④The RAW264.7cells were cultured with10%serum. After9days, the number ofTRAP-positive cells and resorption pits in bone slices were counted.Results①After TBI, the mice showed significant changes in vital signs, including shortapnea, then gradually returned to normal. Cerebral cortical contusion hemorrhage,subarachnoid hemorrhage was observed in brain tissue with naked eyes, simplifiedNSS scale score results show that functions of central nervous system in micechanged significantly after TBI.②Sema3A level in the serum of mice with TBI and femoral fracture was highercompared to the other groups at each time point, and reached the highest level in3days after injury; However, there was no significant difference in OPG of each groupat each time point.③The number of TRAP-positive multinucleated cells and resorption pits wassignificantly different among each groups. It showed that the number of TRAPstaining positive multinucleated cells in Group TBI+F was less than the other groups,and showed significant difference compared with Group F, while there was nosignificant difference between Group TBI+F and Group TBI. Conclusion①the model of mouse with TBI and fracture is successfully established, and it canbe used for the experimental observation of serological changes.②Sema3A in serum from mice with TBI and femur fracture increases significantly.Serum from mice with TBI and femur fractures can inhibit osteoclast precursor cellsdifferentiating into mature osteoclasts. Sema3A may be associated with increasingamount of callus and accelerated fracture healing by inhibiting osteoclastsdifferentiating.